The present invention relates to the treatment of sickle cell disease with N-L-alpha-aspartyl-L-phenylalanine 1-methyl ester.
Under low oxygen tension, sickle cell deoxyhemoglobin (HbS) forms multi-stranded fibers (Rodgers, et al. 1987. Proc Natl Acad Sci USA 84:6157-6161; Eaton, W. A. and Hofrichter, J. 1990. Adv Protein Chem 40:63-279) that force a red blood cell (RBC) into a crescent (sickle) shape (Carache, S. and Davies, S. 1991. Acad Med 66:748-74). In 1949, Pauling et al. demonstrated that HbS was electrophoretically distinct from normal human adult hemoglobin (HbA) and coined the name molecular disease to describe the pathological effects of HbS (Pauling, et al. 1949. Science 110:543-548). Seven years later, Ingram (Ingram, V. M. 1956. Nature 178:792-794) reported that HbS differed from HbA by the substitution of valine for glutamic acid in position 6 of the xcex2 chain. This hydrophobic for polar substitution occurs on the surface of the three-dimensional structure of HbS on the first (A) xcex1-helix (Padlan, E. A. and Love, W. E. 1985. J Biol Chem 260:8280-8291). It creates a sticky site which is covered by a complementary (acceptor) crevice between the E and F helices in the xcex2 chain of an antiparallel Hb molecule in the fibril. Key contact residues in the acceptor site are phenylalanine 85 and leucine 88 from the F helix. Each xcex2 chain thus contains a donor and acceptor site which together interact with two other offset Hb molecules, the key condensation events in producing double stranded helical stacks of indefinite length. As the strands of hemoglobin molecules stack together they, continue to elongate and stretch the normally round, flexible RBC into an inflexible sickle or spiculated shape.
Physiologically, the sickled RBCs impair blood flow, enhance hypoxia and accentuate the production of more sickling (Embury, S. H. 1986. Ann Rev Med 37:361-376). The HbS gene is present in about 8-9% of African Americans (Schneider, et al. 1976. Blood 48:629). If homozygous for the gene, a patient shows the severe symptoms of xe2x80x9csickle cell diseasexe2x80x9d such as anemia, hemolysis, severe muscle pain, thrombotic complications, and even sudden exertional death. A heterozygous individual has xe2x80x9csickle cell traitxe2x80x9d with milder symptoms and more infrequent crises. The gene is believed to have been preserved in successive generations because RBCs containing HbS appear to promote survival in endemic malarial regions of Africa, Asia and European countries on the Mediterranean Sea (Allison, A. C. 1956. Scientific American 195:87-94; Friedman, M. J. and Trager, W. 1981. Scientific American 244:154-164).
Research for therapeutic agents known to delay the onset of sickle cell gelation without introducing unacceptable side effects has been ongoing for many years. (Murayama, M. 1966. Science 153:145-149; Dean, J. and Schechter, A. N. 1978. New Engl J Med 299:804-811; Dean, J. and Schechter, A. N. 1978. New Engl J Med 299:863-870; Dean, J. and Schechter, A. N. 1978. New Engl J Med 299:752-763). No significant approach has been advanced for the treatment of sickling phenomena (Ranney, H. M. 1972. Blood 39:433-439; Aluoch, J. R. 1984. Trop Geogr Med 36:SI-26; Serjeant, G. R. 1997. Br J Haematol 97:253-255; Olivieri, N. F. and Vichinsky, E. P. 1998. J Pediatr Hematol Oncol 20:26-31), although there are many stimulating research approaches and hydroxyurea has some effect. (Dickerson, R. E. and Geis, I. Hemoglobin: Structure, Function, Evolution, and Pathology. Benjamin/Cummings Publishing Co., Menlo Park, 1st Park, ed., 1983). Clinical management of a sickle-cell crisis is usually described as supportive, using fluids for hydration (Scott-Conner, R. E. and Brunson, C. D. 1994. Am J Surg 168:268-274), oxygen for alleviation of hypoxic sickling and analgesics for pain relief (Pollack, et al. 1991. J Emerg Med 9:445-452). Though often effective, even exchange transfusion remains controversial as preventive therapy (Selekman, J. 1993. Pediatr Nurs 19:600-605).
U.S. Pat. No. 5,654,334 discloses APM as a pain reliever which is especially effective in relieving pain associated with osteoarthritis and multiple sclerosis. Further, International Application WO 97/00692 discloses APM as an antipyretic. In a clinical trial, APM was demonstrated to alleviate the pain and inflammation of osteo- and mixed osteo- and rheumatoid arthritis by an unknown mechanism (Edmundson, A. B. and Manion, C. V. 1998. Clin Pharm Therap 63:580-593). Additionally, International Application WO 98/13062 discloses the efficacy of APM in the treatment of a disease affected by the presence of TNFxcex1, particularly arthritis and rheumatoid arthritis. U.S. Pat. No. 5,629,285 discloses sickle cell anemia as one of numerous diseases in which the overproduction or unregulated production of TNFxcex1 has been implicated
It has now been found that APM displays binding behavior with HbS resulting in a modified HbS molecule useful for treatment of the sickle cell family of diseases.

The present invention relates to the use of the compound
where R is CH3 or an alkyl which allows transport of the compound across the red blood cell membrane to prepare a pharmaceutical composition useful for effecting a reduction in sickle cells in a mammal. The preferred compound used to prepare the pharmaceutical composition is N-L-alpha-aspartyl-L-phenylalanine 1-methyl ester (R=CH3). In one embodiment, these compounds are used to prepare a pharmaceutical composition useful for treating sickle cell disorders.
In another aspect the present invention relates to a pharmaceutical preparation in dosage unit form adapted for administration to obtain an antisickling effect in red blood cells, comprising, per dosage unit, an antisickling effective non-toxic amount of a compound comprising 
where R is CH3 or an alkyl which allows transport of the compound across the red blood cell membrane and a pharmaceutical carrier. Preferably, the compound comprises N-L-alpha-aspartyl-L-phenylalanine 1-methyl ester (R=CH3).
In another aspect, the present invention relates to a pharmaceutic dosage form for use as an antisickling agent comprising the compound 
where R is CH3 or an alkyl which allows transport of the compound across the red blood cell membrane, wherein the dosage form comprises preferably from about 1.5 milligrams to about 6 milligrams per kilogram body weight of the compound. More preferably, the dosage form comprises about 6 milligrams per kilogram body weight of the compound, preferably to be administered twice a day. The preferred dosage form comprises N-L-alpha-aspartyl-L-phenylalanine 1-methyl ester (R=CH3).
In another aspect, the present invention relates to a pharmaceutical dosage form comprising an active antisickling ingredient, wherein the active antisickling ingredient comprises the compound 
where R is CH3 or an alkyl which allows transport of the compound across the red blood cell membrane. The effective amount of the compound in the active ingredient is preferably from about 1.5 milligrams to about 6 per kilogram body weight. More preferably, the effective amount of the compound in the active ingredient is about 6 milligrams per kilogram body weight, preferably to be administered twice a day. The preferred active ingredient comprises N-L-alpha-aspartyl-L-phenylalanine 1-methyl ester (R=CH3).
In another aspect, the present invention relates to the use of the compound 
where R is CH3 or an alkyl which allows transport of the compound across the red blood cell membrane to produce an antisickling effect in red blood cells in vitro. The preferred effective amount of the compound is from about 1 milligrams to about 2 milligrams per milliliter. Preferably, the compound is N-L-alpha-aspartyl-L-phenylalanine 1-methyl ester (R=CH3).
In yet another aspect, the present invention relates to a formulation for treatment of red blood cells suspected to contain sickle cells, the formulation comprising the compound 
where R is CH3 or an alkyl which allows transport of the compound across the red blood cell membrane and calcium. Preferably, the formulation comprises N-L-alpha-aspartyl-L-phenylalanine 1-methyl ester (R=CH3).
In yet another aspect, the present invention relates to a method for reducing the number of sickle cells relative to the number of normal red blood cells in a patient blood sample from the time of collection of the blood sample from the patient to a second time of laboratory analysis, comprising the steps of: (a) collecting a blood sample from a patient having a sickle cell disorder, wherein red blood cells in the blood sample have a predisposition to sickle; and (b) adding to the blood sample at the time of collection an effective amount of a composition comprising the compound 
where R is CH3 or an alkyl which allows transport of the compound across the red blood cell membrane, wherein the effective amount causes a reduction in the number of sickle cells relative to the number of normal red blood cells in a patient blood sample at the time of laboratory analysis. Preferably, the compound in the composition is N-L-alpha-aspartyl-L-phenylalanine 1-methyl ester (R=CH3).
In yet another aspect, the present invention relates to a method of treatment of sickle cell disease in a patient by administration of an effective amount of a composition comprising the compound 
where R is CH3 or an alkyl which allows transport of the compound across the red blood cell membrane, wherein the treatment results in a reduction of the number of sickle cells relative to the number of normal cells in the patient""s blood. A preferred effective amount of the compound is from about 1 milligrams to about 6 milligrams kilograms body weight. A more preferred effective amount of the compound is about 6 milligrams of per kilograms body weight. Preferably, the compound in the composition is N-L-alpha-aspartyl-L-phenylalanine 1-methyl ester (R=CH3). The treatment is preferably administered daily. Preferably, the treatment is administered orally.